Research in human chorionic gonadotropin (hCG) relates to three elements. A) structure of hCG protein chains and carbohydrates; B) biology of hCG in fertility and cancer; and C) vaccination strategies for immune targeting of hCG, including against hCG peptides, beta chain, or carbohydrates to generate either humoral or T cell-mediated immune responses.
Human chorionic gonadotropin is a 38 kD heterodimeric glycoprotein. Morgan et al., J. Biol. Chem. 250: 5247, 1975; Hearn and Gomme, J. Mol. Recognit. 13: 223, 2000. Key features of hCG's structure can be seen on diagrams of the primary structure of hCG's alpha and beta chains, as depicted in FIG. 1A. Birken, et al., Clin Chem 49: 144, 2003. The alpha subunit of hCG (alpha-hCG, or hCGα in FIG. 1A) is common to glycoprotein hormones including follicle stimulating hormone (FSH), luteinizing hormone (LH), and thyroid stimulating hormone (TSH). Alpha-hCG's protein chain contains 92 amino acids and carries two N-linked oligosaccarides at residues 52 and 78. hCG's hormone-specific beta chain (beta-hCG, or hCGβ in FIG. 1A) contains 145 amino acids. Relative to the highly homologous LH beta chain and unique among the glycoprotein hormones, beta-hCG has an additional 31 amino acids at the carboxyl terminus. 4 Morgan et al., J. Biol. Chem. 250: 5247, 1975. This carboxyl terminal peptide (CTP) is both kinky and hydrophilic with nine proline (29 mole %) and eight serine (26 mole %) residues. Beta-hCG carries two N-linked oligosaccarides at residues 13 and 30, as well as four O-linked oligosaccharides at residues 121, 127, 132, and 138.
Tertiary structure of the hCG heterodimer is notable for membership in the cystine knot growth factor family (CKGF) of cytokines. Lapthorn et al., Nature 369: 455, 1994; Wu et al., Structure 2: 545, 1994. The CKGF family includes glycoprotein hormones, nerve growth factor (NGF), platelet derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta), among at least forty other such proteins. Hearn and Gomme, J. Mol. Recognit. 13: 223, 2000. CKGF cytokines are characterized by strong, specific, non-covalent dimerization of two subunits. Each subunit features a remarkable, conserved configuration (knot) of three cystine disulfide bonds in which two disulfides form a ring through which the third disulfide bond passes. Secondary structure is primarily of beta strands. Tertiary structure of each subunit is highly elongated with a high surface:volume ratio and absence of any defined hydrophobic core region. Quaternary structure of hCG comprises head to tail association of subunits along their long axes, involving approximately 25% of their surface area. Dimerization is stabilized by a 21 amino acid loop that extends from the cystine knot of beta-hCG and loops around alpha-hCG, forming a disulfide bonded “seat belt”. Beta-hCG may be proteolytically nicked between residues 44 and 45 or 47 and 48 (hCGβn in FIG. 1A). Nicking leads to deactivation of hCG and hastens dissociation of subunits. Cole et al., J. Clin. Edocrinol. Metab. 76: 704, 1993. A urinary metabolite, the core fragment of beta-hCG (hCGβcf in FIG. 1A), has no known function. Norman et al., J. Endocrinol. 164: 299, 2000; Birken et al., Arch. Med. Res. 32: 635, 2001. In addition to the alpha-beta heterodimer, beta-hCG has been found in both monomeric and homodimeric forms. Butler et al., J. Mol. Endocrinol. 22: 185, 1999.
Eight oligosaccharides comprise about 30% of hCG's molecular weight. This is more carbohydrate than found on the closest homolog; LH carries only three N-linked oligosaccharides, two on the alpha and one on the beta chain. Each oligosaccharide carries up to two negatively charged terminal sugars. Thus hCG carries a noteworthy net negative charge. Oligosaccharides associated with hCG are highly heterogeneous, accounting for a substantial proportion of the hormone's size and charge heterogeneity.
Alpha-beta heterodimeric hCG binds to and activates the LH/hCG receptor. Ascoli et al., Endocr. Rev. 23: 141, 2002. By contrast, none of alpha-hCG, beta-hCG, nicked beta-hCG, or beta-hCG core fragment bind to a recombinant human LH-hCG receptor. Ho et al., Early Pregnancy 3: 204, 1997. The carboxyl terminal peptide does not appear to be of any importance to receptor binding or signaling since antibodies specific for this region of beta-hCG do not interfere with LH/hCG receptor signaling. Iverson et al., Curr. Opin. Mol. Ther. 5: 156, 2003; Dirnhofer et al., FAEB J. 7: 1381, 1993. Chemically deglycosylated hCG binds to but does not activate the rat LH/hCG receptor. Chen et al., J. Biol. Chem. 257: 14446, 1982. Individual N-linked carbohydrate moieties likely do not affect hCG function. Hearn and Gomme, J. Mol. Recognit. 13: 223, 2000.
Human chorionic gonadotropin has a demonstrated role in reproduction. Ascheim and Zondek, Klin. Wochenschr 248, 1927. hCG is obligately required for reproduction and appears to have myriad roles in pregnancy given the expression in many tissues of LH/hCG receptors. Rao, Semin. Reprod. Med. 19: 7, 2001; Filicori et al., Fertil. Steril 84: 275, 2005. Some of these proposed receptor-mediated roles include facilitation of cytotrophoblast invasion, angiogenesis, and immunosuppression (Islami et al., Semin. Reprod. Med. 19: 49, 2001; Licht et al., Semin. Reprod. Med. 19: 37, 2001), as well as inhibition of apoptosis. Kuroda et al., Int. J. Cancer 91: 309, 2001. In addition, the net negative charge conferred by extensive sialylation of hCG on the syncitiotrophoblast surface could by itself also be immunosuppressive. Van et al., Int. J. Cancer 38: 915, 1986.
In non-pregnant states, serum hCG may be present at low concentrations via pulsatile secretion of scant quantities from the anterior pituitary. Birken et al., Endocrinology 137: 1402, 1996. Yet hCG is also produced by cancer cells of many non-reproductive tissues. Cosgrove et al., Biochim. Biophys. Acta. 1007: 44, 1989; Stenman et al., Clin. Biochem. 37: 549, 2004. Given the parallels between human reproduction and malignant transformation, hCG has thus been proposed to be a marker of malignant transformation. Acevedo, J. Exp. Ther. Oncol. 2: 133, 2002; Murray and Lessey, Semin. Reprod. Endocrinol. 17: 275, 1999. Consistent with this, alpha-beta heterodimeric hCG has been shown to block cisplatin-induced apoptosis in ovarian carcinoma cells that express the LH/hCG receptor. Kuroda et al., Int. J. Cancer 76: 571, 1998. However, hCG is neither sensitive nor specific for malignancy.
Two surprising observations have been made concerning hCG's putative role in cancer. First, membrane-bound hCG was found on the surface of many different types of cultured cancer cells. Acevedo et al., Cancer 69: 1829, 1992. This was noteworthy because hCG is a secreted protein with no transmembrane domain. Second, serum beta-hCG was noted to be associated with more aggressive, metastatic presentations of bladder cancer. Iles et al., Br. J. Urol. 64: 241, 1989. Also metastatic phenotype was found to correlate with expression of beta-hCG in an animal model. Acevedo and Hartsock, Cancer 78: 2388, 1996. These findings were of uncertain significance because beta-hCG does not bind to the LH/hCG receptor. Subsequently, hCG-beta has been found by multivariate analysis to be an independent negative prognostic indicator in six different epithelial cancers including colorectal, gastric, oral, pancreatic, ovarian, and renal cell. Louhimo et al., Int. J. Cancer 101: 545, 2002; Louhimo et al., Int. J. Cancer 111: 929, 2004; Hedstrom et al., Int. J. Cancer 84: 525, 1999; Louhimo et al., Oncology 66: 126, 2004; Vartianinen et al., Int. J. Cancer 95: 313, 2001; Hotakainen et al., Br. J. Cancer 86: 185, 2002. Curiously, while tissue levels of beta-hCG by immunohistochemistry were also negatively prognostic in colorectal cancer, only serum beta-hCG was significantly prognostic in renal cell carcinoma. Lundin et al., Int. J. Cancer 95: 18, 2001; Hotakainen et al., Int. J. Cancer 104: 631, 2003. Thus soluble beta-hCG can now be presumed to play an important role in cancer progression.
Beta-hCG was found to inhibit apoptosis of bladder cancer cells in vitro as either a monomer or homodimer. Butler et al., Br. J. Cancer 82: 1553, 2000; Butler and Iles, Tumour Biol. 25: 18, 2004. The authors propose a mechanism in which beta-hCG blocks apoptosis mediated by TGF-beta via binding to without activating TGF-beta receptors. Similarly, one can imagine that beta-hCG could inhibit activity of another CKGF cytokine, PDGF. Pietras et al., Cancer Res. 62: 5476, 2002. Engineered expression of beta-hCG in prostate cancer cells has been shown to down-regulate E-cadherin and upregulate invasiveness. Wu and Walker, Cancer 106: 68, 2006. In the latter experiments, conditioned medium was found to confer the same effect, indicating that soluble beta-hCG in the culture supernatant produced this effect. Although the receptor in this instance is unknown, there are at least 40 CKGF cytokine family members (Hearn and Gomme, J. Mol. Recognit. 13: 223, 2000), so involvement of additional, as yet unidentified receptor(s) is likely. The finding that hCG has been shown to inhibit Kaposi's sarcoma has not been thought to be mutually exclusive with hCG's role in diverse epithelial cancers of broader public health significance. Butler and Iles, Clin Cancer Res. 9: 4666, 2003.
Vaccination targeting hCG to regulate fertility has been pursued for decades. Naz et al., Hum. Reprod. 20: 3271, 2005. At the outset these vaccines sought to generate active specific humoral immunity either to the CTP of beta-hCG or to full-length beta-hCG. Lee et al., Mol. Immunol. 17: 749, 1980; Talwar et al., Proc. Natl. Acad. Sci. U.S.A. 91: 8532, 1994. Efficacy of such vaccines has in principle been demonstrated. Stevens et al., Fertil. Steril. 36: 98, 1981; Talwar et al., Proc. Natl. Acad. Sci. U.S.A. 91: 8532, 1994. Embellishments of these approaches have employed different beta-hCG components or recombinant antigen expression as fusion proteins. Rock et al., Vaccine 14: 1560, 1996; Rout and Vrati, Vaccine 15: 1503, 1997; Xu et al., Sheng Wu Gong. Cheng. Xue. Bao. 20: 49, 2004; Yankai et al., Biochem. Biophys. Res. Commun. 345: 1365, 2006; Geissler et al., Lab Invest. 76: 859, 1997.
Similar approaches have been pursued for cancer treatment. Triozzi and Stevens, Oncol. Res. 6: 7, 1999; Moulton et al., Clin. Cancer Res. 8: 2044, 2002. Additional refinements have included passively administered antibodies (Butler et al., Oncol. Res. 14: 93, 2003), monoclonal antibodies specific for the CTP (Kalantarov and Acevedo, Cancer 83: 783, 1998), genetic immunization (Geissler et al., Lab Invest. 76: 859, 1997), and a targeted fusion protein to generate active T cell mediated immunity against beta-hCG. He et al., Clin. Cancer Res. 10: 1920, 2004. A further alternative seeks to target via a monoclonal antibody the O-linked core 2 sugar-containing oligosaccharide isoforms displayed on the CTP of hyperglycosylated hCG (H-hCG). Birken et al., Arch. Med. Res. 32: 635, 2001; Birken et al., Endocrine 10: 137, 1999; Birken, Tumour Biol. 26: 131, 2005; Cole et al., Gynecol. Oncol. 102: 145, 2006; U.S. Pat. No. 6,764,680; U.S. Patent Application No. 2005/0260196.
Most of the above approaches employ an active immunization strategy. Thus months are required for either antibody or T cell mediated immunity to develop, and some recipients will fail to generate an adequate immune response. None of the above approaches to immune targeting of beta-hCG or H-hCG seeks explicitly to block binding of beta-hCG to the LH/hCG or any other receptor. In particular, formulations targeting either the CTP or H-hCG don't block receptor binding. Although experimental methods have been developed to allow targeting of cancer cells that bear surface-bound beta-hCG, the weight of data on beta-hCG's prognostic significance argues that blockade of serum beta-hCG binding to receptors mediating deleterious effects will be critically important in treatment of cancer by immune targeting of hCG. Furthermore, none of the above approaches have been shown to synergize with cancer chemotherapy.
Thus a need exists in the art to generate more effective treatment of cancers that secrete beta-hCG. To address this problem, means are needed to target beta-hCG in a manner that fulfills the following two criteria. First, the agent generated should be able to target serum beta-hCG quickly following passive administration. In practice this could be accomplished by use of monoclonal antibodies or similar mediators of immune specificity. Second, the method must generate treatment that blocks binding of beta-hCG to its receptor(s) mediating deleterious effects associated with cancer progression. In practice this implies generation of immune specificity for an epitope that is both conformationally defined and surface-accessible.